Purification and structural characterization of a cartilage matrix protein.

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.     

Purification, characterization, structural analysis and protein chemistry of a buffalo heart galectin-1

A soluble β-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for β-d-lactose, and 87–92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV–VIS, fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H₂O₂, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained in wet lab.     

Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis.

We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

beta-actinin, a regulatory protein of muscle. Purification, characterization and function.

beta-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. beta-Actinin consisted of two subunits, beta I and betaII, with chain weights of 37,000 and 34,000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. beta-Actinin had the following effects on actin: (1) inhibition of reassociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) acceleration of polymerization of G-actin. All these actions of beta-actinin can be explained in terms of action as an "ending factor". Experimental evidence favored the view that beta-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of polymerization. Fluorescence-labeled anti-beta-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that beta-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that beta-actinin functions as an ending factor for actin filaments.     

Gonococcal protein III. Purification and chemical characterization of the protein,and the DNA sequence of the structural gene

We have purified protein III (PIII) from several strains of gonococcus by extractions with Zwittergent 3,14 followed by cation exchange chromatography and gel filtration. The pI of 8.6 determined by isoelectric focusing was in keeping with the high content of basic amino acids found. PIII from two strains had identical N-terminal sequence. In contrast to PIII in vivo, purified PIII was highly susceptible to proteolysis. Rabit antibodies raised with purified antigen reacted with PIII of all strains tested as well as meningococcal protein 4. Furthermore, intact gonococci or meningococci could absorb 80% of antibodies raised by immunization with the purified PIII. The structural gene of PIII was cloned and the DNA sequenced. The predicted primary structure is strongly homologous to the OmpA proteins of Enterobacteria.     

Purification, characterization and structural analysis of an abundant beta-1,3-glucanase from banana fruit.

An abundant, catalytically active beta-1,3-endoglucanase (EC 3.2.1. 39) has been isolated from the pulp of ripe bananas. Biochemical analysis of the purified protein, molecular modelling, and molecular cloning of the corresponding gene indicate that this banana enzyme closely resembles previously characterized plant beta-glucanases with respect to its amino-acid sequence, structure and biological activity. The results described in this paper demonstrate both the occurrence of an abundant active beta-1,3-endoglucanases in fruits and also readdress the question of the possible involvement of these enzymes in the ripening and/or softening process.     

Purification and characterization of a novel plant metalloproteinase

A novel enzyme, the first metalloproteinase purified from a monocotyledonous plant, was extracted from the endosperm of sorghum seedlings and purified to homogeneity by ion exchange chromatography and size exclusion chromatography. SDS-PAGE analysis reveals a dimeric 17-kDa protein with two 8-kDa subunits linked by disulfide bond(s). The enzyme is 97% inhibited by 1 mM EDTA and is unaffected by inhibitors of aspartic, cysteine, and serine proteinases. Its pH optimum is 7.0 with hemoglobin as substrate.     

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase.

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.     

Evidence for an endogenous ATPase inhibitor protein in plant mitochondria. Purification and characterization

An endogenous ATPase inhibitor protein has been identified and isolated for the first time from plant mitochondria. The inhibitor protein was isolated from potato (Solanum tuberosum) tuber mitochondria and purified to homogeneity. The isolated inhibitor is a heat-stable, trypsin-sensitive, basic protein, with a molecular mass approximately 8.3 kDa. Amino acid analysis reveals a high content of glutamic acid, lysine and arginine and the absence of proline; threonine and leucine. The interaction of the inhibitor with F1-ATPase requires the presence of Mg2(+)-ATP in the incubation medium. The ATPase activity of isolated F1 is inhibited to 50% in the presence of 14 micrograms inhibitor/mg F1. A stoichiometry of 1.3 mol inhibitor/mol F1 for complete inhibition can be calculated from this value. The potato ATPase inhibitor is also a potent inhibitor of the ATPase activity of the isolated yeast F1. The inhibitor resembles the ATPase inhibitors of yeast and mammalian mitochondria, and does not seem to be related to the inhibitory peptide, epsilon subunit, of chloroplast ATPase.     

Purification and structural characterization of polybrominated biphenyl congeners.

Two sets of taurine receptors on rat heart sarcolemma have been identified. The high affinity taurine receptors (Kd=3.5×10^?4M) show a non-cooperative binding profile while the low affinity taurine receptors exhibit positive cooperativity. Taurine binding to the membrane exhibits a typical bell shaped pH profile with maximum binding occurring at pH 8.0. The maximum temperature for binding is 24°C. The effect of various taurine analogues on the receptors was investigated. It was found that binding is prevented by hypotaurine and inhibited to a lesser degree by isethionic acid and cysteine sulfinic acid, while β-alanine was found to increase taurine binding. The effect of several hydrolytic enzymes was also examined and it was shown that several proteases and phospholipase C inhibit binding. The results indicate that the taurine receptors are membrane bound proteins in a phospholipid environment.     

Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Purification and characterization of a proline-rich secretory protein that is a precursor to a structural protein of an insect spermatophore

The spermatophore or sperm sac of Tenebrio molitor (yellow mealworm beetle) is an acellular structure composed mostly of structural proteins, termed spermatophorins. The proteins are derived from the bean-shaped accessory reproductive glands of the male and are assembled into the multilayered structure within the ejaculatory duct. Homogenates of the secretory plug from this gland were used as immunogens for the production of monoclonal antibodies, including one identified as PL 21.1 which recognizes an antigen in the gland and the spermatophore. With the aid of gel filtration and immunoaffinity chromatography with a PL 21.1, we isolated a glandular secretory protein that is a precursor to a spermatophorin with similar electrophoretic mobility. On native polyacrylamide gels, the antigen from gland homogenates has an apparent molecular mass of 370 kDa. On sodium dodecyl sulfate gels, the antigen from the gland and that from the spermatophore have apparent molecular masses of 23 kDa. According to immunoblots of sodium dodecyl sulfate gels, the 23-kDa glandular antigen is organ-specific and adult-specific. By immunocytochemistry with PL 21.1, we found the antigens to be restricted to secretory vesicles of only one cell type in the gland and to a discrete layer in the outer wall of the spermatophore. The 23-kDa secretory antigen is distinguished by being high in glutamic acid/glutamine (15.4%) and in proline (25.2%).     

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.     

Purification and partial characterization of fibrillin, a cysteine-rich structural component of connective tissue microfibrils.

Fibrillin, a connective tissue macromolecule (Mr = 350,000) which is normally insoluble in its tissue form, has been purified from the medium of human skin fibroblast and ligament cells in culture. Analysis of the amino acid composition indicates that fibrillin contains approximately 14% cysteine, of which one-third appears to be in the free reactive sulfhydryl form. Electron microscopic images of fibrillin reveal an extended, flexible molecule approximately 148 nm long and 2.2 nm wide. These length measurements are consistent with shape calculations based upon velocity sedimentation data. It is likely that the material we have purified from cell culture medium represents monomeric fibrillin consisting of a single polypeptide chain. Additional ultrastructural immunohistochemical data presented here suggest a model for the parallel, head-to-tail alignment of fibrillin molecules in microfibrils.     

The 19-kDa glycoprotein coded by region E3 of adenovirus. Purification, characterization, and structural analysis

The 19-kDa glycoprotein (gp 19K) coded by early region E3 of adenovirus is of interest as a model for glycoprotein processing and sorting, as well as for the interaction between viral antigens and class I transplantation antigens. In this paper, we show that gp 19K is a major protein synthesized during early stages of infection of human KB cells. We report the purification of gp 19K to near homogeneity, the preparation of a gp 19K antiserum, and structural analyses on the protein. We have determined the DNA sequence of the gp 19K gene in adenovirus type 5 (Ad5) for comparison with the published sequence (Hérissé, J., Courtois, G., and Galibert, F. (1980) Nucleic Acids Res. 8, 2173-2192) of adenovirus type 2 (Ad2). Fragments produced by cyanogen bromide cleavage of Ad2 gp 19K are in accord with the DNA sequence, as are synthetic peptide antibodies targeted to the NH2 terminus of Ad2 gp 19K and the COOH terminus of Ad5 gp 19K. The Ad2 and Ad5 proteins are quite homologous. Conserved features include an NH2-terminal signal sequence, two potential Asn-linked glycosylation sites, and a 20-residue putative transmembrane hydrophobic domain followed by a 15-residue polar domain at the COOH terminus. We show that cleavage of the signal peptide occurs between the 17th and 18th amino acids on both the Ad2 and Ad5 versions of gp 19K and that both potential sites are glycosylated with exclusively high-mannose (as opposed to complex) oligosaccharides. Secondary structure predictions suggest six alpha-helix regions including the signal peptide and transmembrane domain, two or three beta-sheet regions, and about eight beta-turns including the two glycosylation sites and the regions flanking the transmembrane domain.     

Purification and characterization of a protein phosphatase from winged bean.

A protein phosphatase (WbPP) has been purified from the soluble fraction of the winged bean (Psophocarpus tetragonolobus) shoot extract. The preparation is essentially homogenous as shown by the constant specific activity of the enzyme across the peak fractions, eluted from the thiophosphorylated histone-Sepharose affinity column, the last step of purification and by single protein bands on polyacrylamide gel electrophoresis (PAGE) in the presence as well as absence of denaturating agents. The monomeric nature of WbPP is revealed by an M(r) of 92,000 and 85,000, respectively, as estimated by SDS-PAGE and gel permeation chromatography under non-denaturating conditions. Autophosphorylated calmodulin-like domain protein kinase (P-WbCDPKI) [Saha, P., & Singh, M. (1995). Biochem. J., 305, 205] and phosphohistone H1 (P-hisH1), prepared by using the other homologous CDPK, i.e. WbCDPKII [Ganguly, S., & Singh, M. (1998). Phytochemistry, 48(1), 61], are good substrates of the purified enzyme, while P-hisH1 and phosphocasein prepared by using heterologous cAMP-dependent protein kinase, are respectively very poor and totally inactive as substrate. WbPP is adjudged to be a protein phosphoserine phosphatase since phosphoserine is the only phosphorylated amino acid residue detected in our earlier analysis of P-WbCDPKI and P-hisH1. The enzyme is strongly stimulated by a combination of Mg2+ and Ca2+, without being dependent on either of them and is also unaffected by calmodulin and fluphenazine. Orthovanadate strongly inhibits the enzyme while okadaic acid is a poor inhibitor.     

Purification and characterization of a cholesterol-binding protein from human pancreas.

The protein composition of human intestinal lavage fluids was analysed by electroimmunoassay. In addition to secretory immunoglobulin A and other components that were antigenically related to serum proteins, a number of gut-specific proteins were detected. One of these was found to exhibit the capacity of binding sodium deoxycholate and cholesterol. After isolation of this cholesterol-binding protein from intestinal fluids, immunohistochemical studies utilizing a specific antiserum indicated the pancreas to be the organ of its synthesis. The protein was subsequently purified from necrobiotic pancreas tissues and was found to be composed of a single polypeptide chain with a mol. wt. of 28 000 and an isoelectric point of pH4.9. The deoxycholate binding capacity determined by gel chromatography in the presence of [3H]deoxycholate was calculated to be approx. 24 mol of deoxycholate/mol of protein. In the intestinal fluids the protein appeared to be present in firm association with cholesterol, phospholipids, triacylglycerols and bile salts as a macromolecular protein-lipid complex. The possibility is raised that the pancreas-derived, cholesterol-binding protein may fulfil a function as an intestinal 'lipoprotein'.     

Purification and characterization of a polyhook protein from Caulobacter crescentus.

A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.     

Mavicyanin, a blue copper protein from Cucurbita pepo medullosa. Purification and characterization.

1. The copper protein mavicyanin has been isolated and purified from the green squash Cucurbita pepo medullosa. 2. Mavicyanin contains one type-1 copper/18000 Mr, which can be characterized by: intense absorption maximum at 600 nm (epsilon = 5000 M-1 cm-1/Cu, A280/A600 = 8.0 +/- 0.5, A600/A403 = 7.0 +/- 0.25, maximum of fluorescence emission at 335 nM. 3. In the oxidized state the copper of mavicyanin is 100% detectable by electron paramagnetic resonance (EPR). Computer simulation of the rhombic EPR signal gives gz = 2.287, gy = 2.077, gx = 2.025, Az = 3.5 mT, Ay = 2.9 mT and Ax = 5.7 mT. 4. Like other simple type-1 copper proteins, such as stellacyanin, azurin or plastocyanin, mavicyanin is readily reduced by hydroquinone or L-ascorbic acid. Its midpoint potential E'm was determined to be + 285 mV. The reduced protein reacts rather slowly with dioxygen, but is rapidly reoxidized by ferricyanide.